Abstract
Although ongoing control strategies are attempting to minimize certain metabolic disorders such as hyperketonemia, factors such as genetics, management, environment, and geography still present a potential challenge to the achievement of the desired results. In light of this, this study was conducted (i) to determine the prevalence of subclinical and clinical ketosis based on the concentration of ketone bodies (BHB) using ELISA Kit, and (ii) to examine a number of epidemiological risk factors related to the conditions. From October, 2020 to the January, 2021. A 80 blood samples were collected in a cross-sectional survey in different areas of Mosul. Data included age, management systems, parity, and origin. At the cut-off point ≥1.2-≤ 1.4 mmol / L, the prevalence of subclinical ketosis was 27.5% and for clinical ketosis it was 5%. Results revealed that the animals aged > 3 years were more at risk for subclinical ketosis compared to animals aged ≤ 3 years RR = 2.6471. Multiparous animals were more at risk compared to the primiparous (two or less births) RR = 2.0652. No significant difference between the animals’ origins RR = 0.6863. Indoor and outdoor system animals were more at risk for subclinical ketosis compared to the indoor system only RR = 2.1389. In conclusion, hyperketonemia is prevalent in Mosul. Certain risk factors should be taking into account when planning control programs. Attention should be paid to diagnosis, management, and control of this disease during the risk stage to avert economic loss to the dairy farmers.
Keywords
Main Subjects
Highlights
Article highlights
1- Subclinical ketosis is usually unnoticed and typically associated with lower milk production, poor reproductive performance, and decreased profitability of dairy farms.
2- Affected animals with Subclinical ketosis are more at risk for other disorders like abomasal displacement, metritis, mastitis, retained placenta, diminished fertility, decreased reproductive efficiency, and weakened immune functions
3- To minimize economic losses from SCK disease, it is highly recommended that there is regular monitoring of metabolic tests for the duration of the transition period.
4- Successful control and subsequent prevention depend highly on early detection of subclinical ketosis in dairy farms, when it can be efficaciously obtained by estimation of blood BHB with a cut-off point between ≥1.2-≤ 1.4 mmol / L, and NEFA >0.4 mmol / L in postpartum period.
Full Text
Subclinical ketosis: Prevalence and some risk factors in cross breed and imported breed dairy cows in Mosul, Iraq
M.H. Ali and S.D. Hassan
Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul, Mosul, Iraq
vetdoc600@gmail.com, 0000-0002-1392-0856
hasanali@uomosul.edu.iq, 0000-0002-3010-501x, Corresponding
Abstract
Although ongoing control strategies are attempting to minimize certain metabolic disorders such as hyperketonemia, factors such as genetics, management, environment, and geography still present a potential challenge to the achievement of the desired results. In light of this, this study was conducted (i) to determine the prevalence of subclinical and clinical ketosis based on the concentration of ketone bodies (BHB) using ELISA Kit, and (ii) to examine a number of epidemiological risk factors related to the conditions. From October, 2020 to the January, 2021. A 80 blood samples were collected in a cross-sectional survey in different areas of Mosul. Data included age, management systems, parity, and origin. At the cut-off point ≥1.2-≤ 1.4 mmol / L, the prevalence of subclinical ketosis was 27.5% and for clinical ketosis it was 5%. Results revealed that the animals aged > 3 years were more at risk for subclinical ketosis compared to animals aged ≤ 3 years RR = 2.6471. Multiparous animals were more at risk compared to the primiparous (two or less births) RR = 2.0652. No significant difference between the animals’ origins RR = 0.6863. Indoor and outdoor system animals were more at risk for subclinical ketosis compared to the indoor system only RR = 2.1389. In conclusion, hyperketonemia is prevalent in Mosul. Certain risk factors should be taking into account when planning control programs. Attention should be paid to diagnosis, management, and control of this disease during the risk stage to avert economic loss to the dairy farmers.
Keywords: Subclinical ketosis, ELISA, Prevalence, Risk factors, Mosul
الخلونمیة تحت السریریة: الانتشار وبعض عوامل الخطورة فی أبقار الحلیب الهجینة والمستوردة فی الموصل, العراق
محسن حسن علی و صدام ظاهر حسن
فرع الطب الباطنی والوقائی، کلیة الطب البیطری، جامعة الموصل، الموصل، العراق
الخلاصة
على الرغم من أن استراتیجیات السیطرة مستمرة للتقلیل من الاضطرابات الأیضیة مثل فرط کیتون الدم، إلا أن بعض العوامل مثل الوراثة والإدارة والبیئة والمنطقة الجغرافیة لا تزال تمثل تحدیًا محتملاً لتحقیق النتائج المرجوة. فی ضوء ذلک، أجریت هذه الدراسة لتحدید مدى انتشار الخلونمیة تحت السریری والسریری اعتمادا على ترکیز أجسام الکیتون نوع بیتا هیدروکسی بیوتیریت باستخدام تقنیة الإنزیم المناعی الممتز، وللتحقق من دور بعض عوامل الخطورة الوبائیة على الانتشار. منذ بدایة تشرین الأول 2020 وحتى نهایة کانون الثانی 2021، تم جمع 80 عینة دم من أبقار حدیثة الولادة فی مسح مقطعی لمناطق مختلفة من الموصل. تضمنت البیانات الوبائیة العمر وأنظمة الإدارة وعدد الولادات والمنشأ. اعتمادًا على نقطة القطع ≥1.2-≤ 1.4 مللی مول / لتر، أظهرت النتائج أن انتشار الخلونمیة تحت السریریة کان 27.5% وبالنسبة الحالة الخلونمیة السریریة، کانت 5%. الحیوانات التی یزید عمرها عن ثلاثة سنوات کانت أکثر عرضة للإصابة بالخلونمیة تحت السریریة مقارنة بالحیوانات التی یبلغ عمرها ≤ 3 سنوات بخطورة نسبیة 2.6471 کانت الحیوانات متعددة الولادة أکثر عرضة للإصابة مقارنة بالحیوانات (ولادتان أو أقل بخطورة نسبیة 2.0652). لم یکن هناک فرق معنوی بین منشأ الحیوانات بخطورة نسبیة 0.6863. الحیوانات ذات نظام التربیة الداخلی والخارجی کانت أکثر عرضة للإصابة مقارنة بنظام التربیة الداخلی فقط بخطورة نسبیة 2.1389 فإن الخلونمیة منتشر فی الموصل. یجب أن تؤخذ عوامل الخطورة فی الاعتبار عند التخطیط لوضع برامج السیطرة. یجب الاهتمام بتشخیص هذا المرض وإدارته ومکافحته خلال مرحلة الخطر لتجنب الخسارة الاقتصادیة لمزارعی الألبان.
Introduction
Ketosis (Hyperketonemia) in dairy cows is a predominant problem faced by the animal husbandry industry worldwide, it is a frequently recurring and costly disorder in ruminants with significant economic implications to farm profitability in terms of substantially decreased milk production, minimizing the insemination rate and incurring additional treatment costs. An increase of ketone bodies in the numerous body fluids accompanied by clinical signs such as loss of appetite, unrepresentative licking, and chewing, rapid body weight loss, and decreased milk yield are referred to as clinical ketosis (CK), while subclinical ketosis (SCK) is evident in an increase of ketone bodies in the blood, plasma, serum, urine and milk beyond the standard range in a cow without detectable signs of disease (1). Most of the dairy cows at the transition period (three weeks before and after calving) are affected by diverse physiological, hormonal, and metabolic changes, besides change in feeding and housing, which in turn significantly increases the incidence of the diseases in this timeframe. During the onset of lactation, most dairy cows have a substantially high requirement for glucose and inadequate adaption in this period can result in a negative energy balance and increased risk for occurrence of ketosis and affect animal health and production. Globally, the literature on the prevalence of SCK ranges between 10% and 40% or from 8.5% to 58.8%, depending on the various diagnostic techniques used and thresholds, with the most prevalence during the first 21 days of calving (2). The common authoritative risk factors (including both the herd and Cow-level risk factors) for ketosis are management, the season, herd size, breed, dystocia, and parity (3). Economically subclinical ketosis has a significant impact on dairy farms, and losses from undiagnosed SCK may exceed losses caused by CK. With subclinical ketosis, performance and production efficiency of animals is affected obviously by increasing the culling rates, reproductive decline, decreased milk yield, metritis, withdrawal insemination index, mastitis, abomasal displacement, and risk of developing CK (4). Several biochemical tests to diagnose ketosis by measuring the ketone bodies, predominantly β-hydroxybutyrate (BHBA) in biological fluids are available. Normal cows have plasma BHBA concentrations less of than 1000μmol/L and cows with clinical ketosis have concentrations often in excess of 2500μmol/L. Measurement of blood B-hydroxybutyrate BHBA levels in serum or plasma with a cut-off point of 1.2 to 1.4 mM is the gold standard test for diagnosis of subclinical ketosis and commonly used to differentiate cows with and without SCK (1,5). Several inexpensive cowside tests are also commercially available, but with lower sensitivity and specificity in comparison with the gold standard test (6,7).
In Mosul, Iraq, the occurrence and risk factors for subclinical ketosis are not known, so, the objectives of this study are formulated to address this issue, and bridge this gap to determine the prevalence of subclinical ketosis in cows in Mosul city, Iraq, by measuring blood BHBA levels in serum using ELISA test, in addition to; to examine a number of epidemiological risk factors related to the prevalence of subclinical ketosis.
Materials and methods
Animals and study location
In total, 80 blood samples were collected from the newly-calved cows in a cross-sectional survey in different areas of Mosul city, Iraq, which included Gogjalee, Shalalat, Bazawayah, Al-Intisar, Julechwan, and Tehrawa, from the beginning of October, 2020 to the end of January, 2021. The ages of the animals ranged from two to 10 years, from different management systems (indoor, indoor and outdoor), parity (primiparous and multiparous) and of different origins: local crossbreed and imported breed. Epidemiological information, clinical signs, and clinical vital parameters (pulse, respiratory rate, rumen movements, and heart rate) were recorded in a preformed clinical card.
Samples
A total of 7 ml of the blood was collected from the middle coccygeal vein using sterile syringes after cleaning the area and sterilizing it with 70% ethyl alcohol in the morning (Between 8-9 A.M.). Blood samples were placed in sterile 10 ml glass tubes without anticoagulant to obtain blood serum for estimating the concentration of BHB in serum. Animals with BHB concentration in serum <1mmol/L were considered as control group.
Laboratory analysis
The concentration of ketone bodies (Beta-hydroxy butyrate BHB) in serum samples was measured with an ELISA kit (from Bioassay Technology Laboratory, Shanghai, China) following the manufacturer’s instructions. The plate was read by an Elisa Reader at 450 nm wavelength. The test was performed at room temperature.
Statistical analysis
The prevalence was calculated using descriptive statistics on Excel program 2010 for Windows 10, and the Relative Risk was analyzed for epidemiological factors (age, parity, origin, management system) using the statistical program Epi-Info V.7.2, and the Significant (P <0.05) differences between affected groups and control group were represented by mean and standard error of mean was calculated using T-Test in SPSS program for Windows (version 21; IBM SPSS, USA). All data with p>0.05 were considered significant (8).
Results
The results of the present study indicated that the prevalence of subclinical ketosis in cows in Mosul was 22/80 (27.5%) depending on the concentration of BHB in the serum by ELISA test at the cut-off point ≥1.2-≤ 1.4 mmol/L, and the prevalence of clinical ketosis was 5% (4/80) at a threshold 2.9 mmol/L (Table 1). The study findings also indicated that 36% and 2% of subclinical and clinical ketosis cases respectively, were diagnosed in the first three weeks of birth, and 5% of cases of clinical ketosis at six weeks after birth.
Table 1: Concentration of BHB in the serum of the cows by ELISA test
|
BHBA mmol/L |
Total No. tested |
No. of Animals |
Percentage (%) |
|
≥1.2 - ≤ 1.4 |
80 |
22 (Sub Clinical Ketosis) |
27.5 |
|
≥2.9 |
4 (Clinical Ketosis) |
5 |
|
|
<1 |
54 ( -ve control group) |
67.5 |
This study showed significant (p <0.03) difference between the affected animals depending on the age. Animals aged > 3 years were more at risk for subclinical ketosis compared to animals aged ≤ 3 years, and with a relative risk (RR=2.6471, CI= 1.34 - 5.21) (Table 2). Findings also indicated a significant (p <0.05) difference between the affected animals depending on the number of births, where the multiparous animals (three and more births) were more at risk for subclinical ketosis compared to the primiparous (two or less births) with a RR=2.0652, CI= 1.04 - 4.04 (Table 2).
The findings indicated that no significant (p <0.4) difference between affected animals, depending on the animals’ origin, as the relative risk between local crossbreed and imported infected animals (RR=0.6863, CI=0.28 - 1.64). The results of this study also indicated a significant (p <0.04) difference between the affected animals depending on the management systems, where the animals with indoor and outdoor systems were more at risk for subclinical ketosis compared to the animals with the indoor system with a relative risk (RR=2.1389, CI= 1.01 - 4.52) (Table 2).
Table 2: Risk factors for subclinical ketosis based on BHB concentration
|
Factors |
No. case tested |
No. of +ve (%) |
RR |
CI |
P |
|
Age |
|
|
|
|
|
|
<4years |
9 |
5 (6.25%) a |
1 |
|
|
|
≥4 years |
71 |
17 (21.25) b |
2.6471 |
1.34 - 5.21 |
0.03 |
|
Parity |
|
|
|
|
|
|
≤ 2 |
23 |
10 (12.5%) a |
1 |
|
|
|
≥ 3 |
57 |
12 (15%) b |
2.0652 |
1.04 - 4.04 |
0.05 |
|
Origin |
|
|
|
|
|
|
Local |
24 |
5 (6.25%) a |
1 |
|
|
|
Imported |
56 |
17 (21.25) a |
0.6863 |
0.28 - 1.64 |
0.4 |
|
Management |
|
|
|
|
|
|
Indoor |
36 |
14 (17.5%) a |
1 |
|
|
|
Indoor and outdoor |
44 |
8 (10%) b |
2.1389 |
1.01 - 4.52 |
0.04 |
Values significantly (P < 0.05) different labeled with different letters (a, b or c). RR (Relative Risk), CI (Confidence interval), P (probability).
Discussion
The current study showed a high prevalence of subclinical ketosis in cows in Mosul, Iraq, the occurrences of subclinical and clinical ketosis were 27.5% and 5%, respectively, depending on the BHBA concentration in serum by ELISA test. The prevalence rate of the current study may agree with or differ from other studies conducted in other countries. In Iran, the prevalence of subclinical ketoacidosis was 14.4% and 18.42% (9,10). In Turkey, it was 33.3% (11). Suthar et al. (4) indicated in a study that included several European countries include Italy, Hungary, Poland, Serbia, Slovenia, Spain, Germany, Portugal and Turkey that the overall occurrence of subclinical ketosis was 21.8%, ranging from 11.2 to 36.6%. Several reasons, such as geographical area, season, number of animals examined, techniques used for diagnosis, management system, breed, the threshold used, and type of sample, could explain the difference in prevalence in this study and other studies in other regions or countries. These findings are in accordance with those recorded by (11-14). In general, numerous studies indicated that the global prevalence rates for subclinical ketosis range from 8-40% (15), and clinical ketosis 2-15% (16).
This study showed that the animals aged > 3 years were significantly more at risk for subclinical ketosis compared to animals ≤ 3 years of age, relative risk (RR = 2.6471). The reason could the multiparity and increased milk production. Or in other hand, it may be due to the fact that young cows may increase metabolic activities during early lactation, against stressor conditions (i.e., NEB). This adaptation and activity moderately decrease with the progress of age. This finding agrees with results of other studies (3,17).
Results also showed that the multiparous animals (three and more births) were significantly more at risk for subclinical ketosis compared to the primiparous (two or less births) with RR = 2.0652. It is possible that the reason is the high milk production, which may be predisposed to negative energy, as the cows in the first or second calving may not reach the peak of milk production in their early stages of completion compared to multiparous cows. This result is in agreement with the outcomes of other studies by (18,19).
The finding of this study indicates that there is no significant difference between the animal’s origin (RR = 0.6863). This result is in agreement with (20). This can be attributed to the absence of a difference in the quantities of milk production between imported and local crossbreed cows in addition to the difference in management and feeding systems and environmental conditions, which are among the critical factors that affect the ability of cows to adapt to the different conditions that exist between different countries. This result is supported by previous studies (14,21). Contrary to the results of this study (7,20) indicated a significant difference in the occurrence of subclinical ketosis between the animal breeds, and the prevalence rate may be high in animals with high genetic potential milk production, as these animals are unable to withstand the pressures of high nutritional requirements for milk production and experience a negative energy phase.
The results of this study revealed significant difference between the affected animals depending on the management systems, whereby the animals exposed to indoor and outdoor systems are more at risk for subclinical ketosis compared to the animals exposed to the indoor system only (RR = 2.1389). The results of the present study are in agreement with (22). This could be attributed to the availability of concentrated nutrition for animals raised by the indoor feeding system and its reduction in cows raised in the mixed system (indoor and outdoor feeding), as reducing the concentrated diet may decrease the ability of cows to obtain sufficient energy to meet the requirements for milk production and subsequently risk the development of negative energy balance. Yang et al. (23) confirmed that decreasing the feed intake coinciding with the requirement to maintain a high milk production is the cause of ketosis in milk-producing cows.
Conclusions
In conclusion, findings of the current study showed that SCK is prevalent in dairy cows in Mosul, Iraq, and can be observed at various rates in many countries worldwide. The major risk factors for SCK are age, parity, and management system. More epidemiological studies are recommended to evaluate the magnitude of the disease and its economic impact.
Acknowledgements
The authors are grateful to the College of Veterinary Medicine, University of Mosul, for their support and cooperation. The authors wish to thank the laboratory of the veterinary teaching hospital, for their support and the animal’s owners for their cooperation.
Conflict of interest
Authors declare no conflict of interests of the manuscript.
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